Microvascular occlusion by emboli occurs spontaneously throughout life and is common in a variety of disease processes. Although these small vessel occlusions may produce little acute symptomatology, their cumulative effect is likely to lead to organ dysfunction. This is especially relevant in the brain where micro-occlusions may eventually lead to cognitive impairment. The current view is that microvascular emboli are normally cleared by hemodynamic forces and the fibrinolytic system. However, we recently discovered an alternative cellular mechanism that effectively removes from the cerebral microvasculature emboli composed of virtually any substance including those not susceptible to fibrinolysis such as atheromatous cholesterol fragments. Clearance occurs by a previously unknown process of microvascular plasticity involving the engulfment of entire emboli by endothelial membrane projections and their subsequent translocation into the perivascular parenchyma leading to rapid reestablishment of blood flow and vessel sparing. In aging mice, the rate of embolus extravasation is severely delayed. Although the molecular control of the extravasation mechanism is likely to be complex, pathways involved in mechanotransduction, vascular plasticity, cytoskeletal dynamics, remodeling of endothial junctions and extracellular matrix are likely to play a critical role and may be affected in aging. We hypothesize that alterations in these molecular pathways can significantly change the efficiency of embolus extravasation, impacting the viability of occluded microvessels and surrounding tissue. To test this, our research program will investigate the molecular control of the extravasation process by using mutant mice and novel methods for focal vascular drug delivery. The precise effects of these manipulations on the dynamics of endothelial and perivascular cells will be characterized at high spatial and temporal resolution by multicolor two photon imaging in live mice and confocal and transmission electron microscopy in fixed tissues. Furthermore, we will test selected molecules for their ability to rescue the delayed extravasation phenotype in aging. To increase the throughput of our molecular and cellular analysis, we will develop a new model of microvascular embolization in zebrafish and take advantage of its versatility for pharmacological and genetic manipulations as well as in vivo imaging. Finally, we will examine in mice and zebrafish, the consequences of altering the efficiency of the extravasation process on the viability of blood vessels and surrounding parenchyma. Together, these translational experiments will provide a framework for understanding the cellular and molecular basis of this critical mechanism of microvascular clearance while suggesting targets for therapeutic intervention. Our results could have important implications in vascular biology, systemic embolic disorders, stroke and dementia. PUBLIC HEALTH RELEVANCE: Occlusion of microvessels in various organs is likely to occur frequently throughout life. The cumulative effect of these occlusions may lead to organ damage. In the brain, this may be the basis for age related cognitive decline and dementia. We have discovered a physiological mechanism that efficiently eliminates virtually any type of material occluding these small blood vessels. Alterations in the efficiency of this mechanism could have critical implications in a variety of diseases. The goal of our proposal is to develop an innovative approach using molecular manipulations in mice, zebrafish and live microscopy to discover molecular pathways that control this novel mechanism and to determine its importance in salvaging blood vessels and other cells in a variety of organs including the brain.